DK172973B1 - Enhanced emulsions of highly fluorinated organic compounds - Google Patents

Abstract

Improved emulsions of highly fluorinated organic compounds. The emulsions comprise a highly fluorinated organic compound, an oil, that is not substantially surface active and not significantly water soluble, and a surfactant. They are characterized by a well-defined relationship in the relative amounts of the three components.

Description

DK 172973 B1 i

The present invention relates to improved emulsions of highly fluorinated organic compounds as well as to processes for their preparation and use. More particularly, the invention relates to fluorocarbon-containing emulsions with an acceptable particle size distribution both after sterilization and in the presence of serum, which emulsions are safe at high doses and total replacements and exhibit good storage stability at room temperature. These emulsions include a highly fluorinated organic compound, an oil which is not substantially surfactant and not substantially soluble in water, as well as a surfactant. The emulsions are characterized by a well-defined ratio of the relative amounts of these three components. Such emulsions are particularly useful in preparations for use as oxygen transport agents, "artificial blood" or red blood cell replacement, in the treatment of heart attacks, strokes and other vascular obstructions, as adjuvants in coronary balloon angioplasty and in radiation and chemotherapeutic treatment of cancer as well as contrast agents in biological imaging.

It is well known that highly fluorinated organic compounds such as perfluorocarbon compounds ("PFC") are chemically and pharmaceutically inert. They are also 1 capable of dissolving, transporting and delivering oxygen 1 biologically 20 and chemically significant quantities. These properties make them potentially useful as oxygen transport agents, as "artificial blood" or as a replacement for red blood cells, in the treatment of heart attacks, strokes and other vascular obstructions, as adjuvants in coronary angioplasty, in radiation and chemotherapeutic treatment of cancer, and as 25 contrast agents at various biological imaging modalities such as nuclear magnetic resonance, ultrasound, X-ray and positron emission tomography.

However, pure fluorocarbon fluids cannot be injected into the blood stream as their hydrophobic nature makes them immiscible with the blood. As a result, they can cause vascular obstruction and lead to death when transported to small blood vessels. Therefore, for medical applications requiring intravascular injection, such highly fluorinated organic molecules must be dispersed as physiologically acceptable aqueous emulsions. See e.g. L. C. Clark, Jr. et all, "Emulsions of Perfluorinated Sol -35 vents for Intravascular Gas Transport", Fed. Proc., 34 (6), pp. 1468-77 (1975), K. Yokoyama et al., "A Perfluorochemical Emulsion as an Oxygen Carrier", Artif. Organs (Cleve), 8 (1), pp. 34-40 (1984), and U.S. Patents 4,110,474 and 4,187,252.

2 DK 172973 B1

The medical utility of such emulsions of highly fluorinated organic compounds as "artificial blood", as a substitute for red blood cells, as oxygen transport agents or as contrast agents in biological imaging, has so far not been as successful as hoped.

This is because none of the known fluorocarbon-containing emulsions meet all requirements for a preferred "artificial blood" or oxygen transport agent.

These requirements include: 10 (1) Particle size distribution (PSD) after sterilization below 400 nm

A critical parameter for stability and safety is the emulsion's 15 PSD. A comprehensive literature in the field shows that a PSD> 400 nm results in excessive toxicity as the microcirculatory system filters out large particles, leading to a clogging of the microcirculatory vasculature and ultimately to general ischemia. The particle size will also affect the rate at which the emulsion is removed from the circulatory system and emulsions that do not remain in the circulatory system will not be effective as blood substitutes. Thus, since a final heat sterilization of these emulsions is needed to eliminate the possibility of sepsis before use, emulsions exhibiting a PSD> 400 nm (after sterilization) are not acceptable.

(2) Serum stability characterized by a PSD $ 400 nm after five days 1 serum or ionic solutions 30

Of great importance to a given emuis in our formulation is stability to the serum environment. A lack of serum or ionic stability results in particles that grow in vivo, resulting in 1 embolism that can clog the microvasculature and lead to paralysis and death. In addition to the catastrophic growth leading to death, moderate in vivo involvement in cell growth results in faster removal from the circulatory system and thus in reduced efficiency as a replacement for red blood cells or as an oxygen transport agent. Therefore, emulsions exhibiting a PSD> 400 nm (after DK 172973 B1 3 5 days 1 serum or lone solutions) are not acceptable.

(3) High dose survival 5

A major limitation to the usefulness of an emulsion is the IDjq value or the maximum safe dose. Various authorities limit the level of use of an emulsion on the basis of some fraction of the LD ^ value. Emulsions having a higher maximum allowable dose and thus can be used in higher, more effective dosages and within a larger clinical field of application will be more widely accepted by medical science. An acceptable emulsion should have a LD, µg value in rats of at least 16 ml / kg perfluorocarbon component - the active ingredient of the emulsion.

15 (4) Survival at Total Replacements

Total replacement transfusions constitute the most stringent test of the emulsion's safety and effectiveness. In total replacement, the hematocrit of the animal is reduced to 3% or less, a level that would be fatal without intervention. At the same time, the emulsion in question is infused isovoluminously.

Thereafter, the animal's physiological functions, survival, general condition and health, as well as intravascular persistence are checked. Emulsions, which lead to a high level of survival, are believed to exhibit great safety and efficiency. An acceptable emulsion should have a survival rate of at least 70% for total rat replacements.

(5) Storage stability 30

An essential criterion for the commercial utility of an emulsion is its storage stability. Emulsions that cannot be stored for several months either at 4 ° C or more preferably at 25 ° C will not be useful in the art. Their storage stability will be too short in terms of the time difference between manufacture and quarantine, shipping and use.

In addition to these requirements, fluorocarbon-containing emulsions to be used as "artificial blood" or as "substitute for red blood cells" must supply sufficient oxygen to the body's tissues.

As is well known, the oxygen of the blood is usually transported by hemoglobin, a strong specialty in serous protein that absorbs oxygen into the lungs 5 and releases oxygen into the body's tissues. When inhaled and present in the alveoli of the lungs, atmospheric oxygen having an oxygen partial pressure (p02) of about 150 mm Hg, the arterial blood p02 is approx. 100 mm Hg (due to the water vapor that saturates the air in the pulmonary alveoli, the bronchial circulation and other sources of arterial-venous shunting through the lungs).

10 At this relatively low PO 2, all oxygen carrier sites on the hemoglobin are almost completely saturated (97%). Thus, when the oxygen dissolved in the arterial blood plasma is in equilibrium with the hemoglobin in whole blood, 100 ml of this blood will carry approx. 20 ml of oxygen (20% by volume).

As this oxygen-carrying blood moves into the capillaries, where it delivers its oxygen to the tissues, it comes in dynamic oxygen equilibrium with the oxygen present in the pervascular interstitial fluid, which has an average pH of 40 mm Hg. This difference in p02-ni level between the arterial and venous side of the circuit allows a supply of the tissues with their normal needs of approx. 5 ml oxygen per 100 20 ml whole blood (5% by volume).

Unlike hemoglobin, which actually binds and then releases oxygen, emulsions of highly fluorinated organic compounds simply dissolve oxygen. Thus, the amount of oxygen supplied by a fluorine-containing emulsion depends on the difference between the arterial 25 p02 and the venous p02, the solubility of oxygen in the fluorocarbon and the percentage (volume) of the fluorocarbon 1 emulsion.

Supply of O 2 ((arterial pO 2 - venous pO 2) / 760) (% by volume) x (solubility of O 2 in the fluorine compound)

For use as "artificial blood" or red blood cell replacement, a fluorine-containing emulsion should add at least as much oxygen as whole blood - 5 volume percent. Thus, in the 100% O2 inhalation mixture used for up to 12 hours in situations requiring 1n-14 tensive care, such emulsion should contain at least approx. 20% by volume of fluorine compound. For example (using the equation cited above): desired 02-1i loading = 5% by volume 5 arterial pO ^ = 600 mm Hg (100% 02) * venous pO £ = 40 mm Hg solubility of 02 in fluorine compound = 33 10% by volume ** Required by volume of fluorocarbon = x * The approximate, conventionally estimated arterial p02 of a healthy 15-year-old at 100% 0 ^.

** At 37 ° C, most perfluorocarbon-containing compounds have similar Bunsen oxygen solubility coefficients. They are in the range between approx.

0.32 and 0.35, i.e. that the solubility of O 2 therein is between ca. 32 and 35 (volume) percent.

0.05 x = ...................................... 20.5% 1600- 401 (0.33) 760 25 In the 40 to 80% O 2 inhalation mixture which is tolerated for more than 12 hours under hospital conditions, such emulsions should contain from ca. 25 to approx. 60% by volume of fluorocarbon, and most preferably from 30 to 55% by volume.

30 It is clear from the foregoing that without relatively high PFC content, perfluorocarbon emulsions will not be able to supply the quantities of oxygen available from whole blood. In addition to the oxygen supply deficiencies of low volume PFC emulsions, two additional factors are critical for the medical use of perfluorocarbon emulsions. The first is the need for a high oxygen supply per day. volume unit administered fluid. There is a limit to the volume of fluid that can be administered to a patient, especially if the heart or kidneys are thought to be attacked. The other further disadvantage of PFC low volume percent emulsions is that, at high volume transfusers, it is usually necessary to mix the emulsion-administered emulsion with other solutions necessary to meet the patient's ionic and oncotic needs. . A dilution of low% PFC emulsions with "support" solutions containing salts and suitable plasma expansion agents leads to a "dilution" of the already low oxygen delivery capacity of these formulations.

An illustration of the deficiencies of known fluorocarbon containing emulsions is "Fluosol DA 20%", the only fluorocarbon emulsion reached for 10 clinical testing as "artificial blood". It is an approx. 10% by volume emulsion of two fluorocarbons - perfluorodecalln and perfluorotripropylamine - in a mixture of two surfactants - egg biophospholi pi d and "Pluronic F-68". It is not stable in liquid state and must be stored frozen (Yokoyama et al., Supra). In addition, the required presence of the perfluorotripropylamine in this emulsion to aid in "stabilizing" it actually eliminates the medical utility of the emulsion (as artificial blood or oxygen transport agent), since the half-life of the perfluorotripropylamine in the liver and other body tissues is longer than desired. (see, e.g., K. Yokoyama et al., supra). Finally, 20 is this emulsion, since it contains only approx. 10% by volume of fluorocarbon, much less therapeutically effective as "artificial blood" than desired due to its low oxygen supply capacity - approx. 2.4% And at 100% Inhaled Oxygen (see, e.g., "Fluosol-DA As A Red Cell Substitute In Acute Anemia", N.E.

Jour. With. 314, pp. 1653-66 (1986)). This is substantially less than the 5 25% by volume oxygen required to maintain healthy physiological functions.

A further illustration of these deficiencies is the three fluorocarbon emulsions mentioned in Jeppsson et al., "Particle Size Distribution Of A Fluorochemical Emulsion", 1 HS Symposium Research on Perfluoroche- ricals in Medicine and Biology, Huddinge, Sweden, 28 to April 29, 1977, Carolina Institute Research Center, Proceedings, Novakova et al., ed., pp. 108-113 (1978). These emulsions contain approx. 15% by volume of fluorocarbon - too little to be used as "artificial blood" or "red blood cell replacement" - an application that, moreover, has not even been proposed by Jeppsson at any time.

An attempt to solve the problems of these known fluorocarbon-containing emulsions is described in Shaw-Clark's European Patent Application no.

231,091. The emulsions according to this application are characterized by a high fluorocarbon content and good stability at room temperature and after sterilization. They include an oil which is not substantially surfactant and is not substantially soluble in water, a surfactant and a highly fluorinated organic compound. Although they represent a significant improvement over previous emulsions, the Shaw-Clark type emulsions include some emulsions that do not meet all of the additional requirements described above for emulsions preferred for use as "artificial blood", oxygen transport agents, or contrast agents. biological imaging.

Jeppsson (above) also mentions oil, surfactant and fluorocarbon-containing emulsions. See e.g. Jeppsson, supra, and European Patent Applications Nos. 220,152 and Nos. 220,153. Jeppsson does not suggest that these emulsions enable the preferred higher concentration of perfluorocarbons required for "artificial blood", oxygen transport agents, or contrast agents for biological imaging. Jeppson also does not imply that his emulsions meet the other requirements for preferred emulsions for use in these applications. In fact, Jeppsson's emulsions include many emulsions that do not meet all the other requirements set for the preferred emulsions described above.

The present invention solves the above problems.

It provides, for the first time, emulsions of highly fluorinated organic compounds which meet all the requirements of the above-described preferred emulsions.

The emulsions of the invention are characterized by (1) a particle size distribution after sterile Isation of less than 400 nm and preferably of less than 300 nm, (2) a serum stability characterized by a particle size distribution of less than 400 nm and preferably of less than 300 nm. 300 nm after five days in serum or ionic solutions, (3) an LD 50 value in rats of at least 16 ml / kg of fluorocarbon component in the emulsion, (4) a survival rate of at least 70% at total replacement in 1 rat, and (5) a storage stability of at least several months at 4 ° C and preferably at 25 ° C.

The improved emulsions of the invention comprise at least one highly fluorinated organic compound, an oil which is not substantially surfactant and not substantially soluble in water, and a surfactant. More importantly, the emulsions of the invention are characterized by specific and defined ratios of the relative amounts of the three components. The fluorocarbon component is present in the emulsion 1 in an amount of 20 to 60% by volume. The amounts of surfactant and oil depend on the percentage of fluorocarbon volume and are defined by that shown in FIG. 1 and at the surfaces defining this volume.

The invention also encompasses methods and compositions in which these improved emulsions are used as oxygen transport agents, "artificial blood" or red blood cell replacements and as contrast agents in biological imaging, as well as in other known medical compositions and applications.

In the following, the invention will be further elucidated with reference to the drawing.

FIG. Figure 1 is a three-dimensional diagram - w / w% oil / fluorocarbon, w / w% surfactant / fluorocarbon and volume percent fluorocarbon-containing compound - of the improved emulsions of the invention. The FIG. 1 and the surfaces defining the volume describe the improved emulsions of the invention. The filled circles in FIG. 1 represents emulsions according to the invention which meet all the above described requirements of the emulsions. The unfilled circles represent emulsions that do not meet one or more of these requirements.

The emulsions of the invention comprise at least one highly fluorinated organic compound, an oil which is not substantially surfactant and is not substantially soluble in water, and a surfactant.

The emulsions of the invention are stable at room temperature for 25 long periods - at least several months. They are stable against stirring and against mixing with various physiological additives, including, but not limited to, saline, Tyrode solution, Ringer's lactate solution, serum and serum products. They exhibit essentially no phase separation and substantially no change in particle size or droplet distribution during storage. In addition, they allow the use of highly fluorinated organic compounds which exhibit acceptably fast excretion times from the liver and other body tissues. In addition, they allow the use of high fluorocarbon concentrations, resulting in emulsions with the high oxygen delivery capacity required for use as therapeutically effective blood substitutes, oxygen transport agents and contrast agents in biological imaging. Because of their stability, even after sterilization 1, they retain the above-mentioned properties of a conventional laboratory autoclave at 121 ° C for 15 minutes.

DK 172973 B1 9

Among the highly fluorinated organic compounds which can be used in the emulsions and in the methods of the invention are compounds which have previously been termed useful as oxygen transport agents, "artificial blood" or red blood cell replacement 5, and as contrast agents in biological imaging. They include perfluorocarbons, e.g. perfluorodecaline, perfluoroindane, perfluorotrimethylbicyclo [3.3.l] nonane, perfluoromethyladamantane, perfluoro-dimethyladamantane and perfluoro-2,2,4,4-tetramethylpentane, perfluoramines having 9 to 12 carbon atoms, e.g. perfluorotripropylamine, perfluorotributyl-amine, perfluoro-1-azatricyclic amines, bromine or solder substituted fluorocarbons, as well as F-4-methyloctahydroquinolidizine and perfluoroethers.

Such compounds are e.g. disclosed in U.S. Pat.

3,962,439, 3,493,581, 4,110,474, 4,186,253, 4,187,252, 4,252,827, 4,423,077, 4,443,480, 4,534,978 and 4,542,147, EP patent applications no.

15 80,716 and 158,996, GB patent 1,549,038 and DE disclosure no. 2,650,586. Of course, it is to be understood that mixtures of any of these highly fluorinated organic compounds can also be used in the emulsions and in the processes of the invention.

The emulsions of the invention contain one or more perfluorocarbons and more preferably a perfluorocarbon selected from perfluorodecaline, perfluoromethyladamantane, perfluorodimethyladamantane, perfluorooctyl bromide, perfluoro-4-methyloctahydroquinolidizine, perfluoro-N-methyl-N-methyl-N-methyl-N decalin, perfluoro-bicyclo [5.3.0] decane, perfluorooctahydroquinolidizine, perfluoro-5,6-dihydro-5-decene and perfluoro-4,5-dihydro-4-octene. Most preferably, the perfluorocarbon is perfluorodecaline or perfluorooctyl bromide. For use as a contrast agent in biological imaging, perfluorooctyl bromide is the preferred highly fluorinated organic compound of the invention.

While the highly fluorinated organic compounds or a mixture of such compounds may comprise from 20 to 60% by volume of the highly fluorinated organic compound, preferred emulsions of the invention comprise from 30 to 55% by volume of fluorocarbon. Most preferably, the emulsions contain 40% by volume.

Among the non-substantially surfactant and non-water-soluble oils which can be used in the emulsions and in the processes of the invention are liquid fatty oils, hydrocarbons, waxes such as monoesters of a fatty acid and a monohydroxy alcohol, long chain ethers, diglycerides, silicone oils and nitriles. . These include e.g. pal 10 DK 172973 B1 mltoyl oleate, octyl nitri 1, dodecyl nitrile, soybean oil, dye starch oil, hexadecane, diglycerides with a C12 ug carbon chain and mineral oil. As is the case with the fluorine-containing component, these oils can be used individually or in different combinations in the emulsions and in the methods of the invention.

Of course, when the emulsions of the invention are to be used medically, the oil or combination of oils must be physiologically acceptable. When the emulsions of the invention e.g. should be used as "artificial blood" or therapeutically as oxygen transport agents or contrast agents, preferably physiologically acceptable liquid fatty oils such as soybean and color time oil are used.

Among the surfactants that can be used in the emulsions of the invention are any of the known anionic, cationic, nonionic and zwitterionic surfactants. These 15 include e.g. anionic surfactants such as alkyl or aryl sulfates, sulfonates, carboxylates or phosphates, cationic surfactants such as mono-, di-, tri- and tetraalkyl or aryl ammonium salts, nonionic surfactants such as alkyl or aryl Compounds whose hydrophilic moiety consists of polyoxyethylene chains, sugar molecules, polyalcohol derivatives or other hydrophilic groups which may be combinations of the above anionic or cationic moieties and whose hydrophobic moiety consists of any other polymer such as polyisobutylene or polypropylene. Again, combinations of these surfactants can, of course, be used in the emulsions of the invention. In addition, mixtures of compounds, one or more of which are not surfactants, but act as surfactants when combined, may also be useful and useful as a surfactant component of the emulsions of the invention.

Again, when the emulsions of the invention are to be used in "artificial blood" or therapeutically as oxygen transport agents or contrast agents, the surfactants or a combination thereof must be physiologically acceptable. For example, for such applications, nonionic or zwitterionic surfactants are preferred. The surfactants used in the emulsions of the invention are preferably one or more of the following: egg and soybean phosphatides, lecithin and any of the series of BASF Wyandotte formulations. of polyoxyethylene oxides sold under the trade name "Pluronic", in particular "Pluronic F-68". Of course, many other polyethylene oxide-based surfactants can be used, but typically they are not readily available through conventional trade channels.

In addition to the highly fluorinated organic compounds, oils and surfactants, the emulsions of the invention contain water and may also contain or mix with other components conventionally used in "artificial blood" or in red blood cell substitutes, in oxygen transport agents or contrast agents for biological imaging. They include isotonic agents, osmotic pressure regulators, serum stretching agents and antioxidants. For example, they have succeeded in incorporating glycerol as a tonicity-adjusting agent in the preparation of these emulsions to obtain solutions of physiologically acceptable osmolarities. The appropriate amount to obtain isotonicity with respect to whole blood will be apparent to those skilled in the art. In addition, after preparation and heat sterilization, these emulsions have been shown to be admixed with 0.9% saline, Ringer's solution added with lactate, serum and serum products without detrimental effect on the particle size and stability of the emulsion. Other adjuvants may also be used in the emulsions of the invention. Among these are oncotic agents such as dextran, HES and antioxidants.

The emulsions of the invention are characterized by a well-defined right ratio of the relative amounts of the fluorocarbon, oil and surfactant components. This specific relationship or mutual dependence is shown and best described by reference to FIG. First

In FIG. 1, the emulsions of the invention are defined as a volume in a three-dimensional diagram of weight / weight percent oil / fluorocarbon, weight / weight percent surfactant / fluorocarbon and volume percent fluorocarbon. Emulsions falling within this volume, or on the surfaces defining it, fall within the scope of the invention.

Conversely, emulsions which fall outside the described volume and these surfaces are not included in the invention.

Using FIG. 1, the person skilled in the art can thus easily select the range of oils and the range of surfactants that can be used in a given volume percent fluorocarbon to prepare the emulsions of the invention. More preferably, those skilled in the art will be able to select a specified amount of oil and surfactant within the defined range for use in preparing an emulsion of the invention.

Although FIG. 1 is based on emulsions prepared with perfluorodecalin, egg white ommelecin and color time oil, it is believed that other combinations of fluorocarbons, oils and surfactants as described above will exhibit a similar mutual dependence between the components. However, it should be understood that slightly different volumes and surfaces can define the actual ratio of the components of these other 5 emulsions. It is clearly one of ordinary skill in the art to determine these exact volumes and surfaces beyond the scope of the experiment and without departing from the spirit and scope of the invention from the teachings of the present application.

In the more preferred emulsions of the invention, the fluorocarbon is perfluorodecaline, the surfactant is egg yolk ommelecitin and the oil is color-time oil. In the most preferred emulsions according to the invention, the perfluorodecaline is present with approx. 40% by volume, the lecithin is present with approx. 2.3 wt / wt% lecithin / PFD and the color time oil are present with approx. 2.6 wt / wt% oil / PFD.

The emulsions of the invention can be prepared using any order of mixing of the three components and water. However, it is preferred to first mix the oil with an aqueous dispersion of the surfactant. Next, the final emulsion is prepared by adding the fluorocarbon.

Mixing and emulsification of the components of the emulsion according to the invention can be carried out with any of the commercially used mechanical homogenizers and does not require the use of advanced equipment such as ultrasonic homogenizers, although such devices can be used for laboratory scale production. It is preferred to use an inert atmosphere to prevent degradation of the surfactant and fatty oils, and to use temperatures preferably between ca. 45 and 55 ° 0 to reduce the viscosity of the emulsified material.

The following examples are intended to illustrate only and not to limit the invention.

examples

Methods 35

Preparation of Vandio Lecithin Dispersion

Powdered, refined egg tissue surrounding ecitin was obtained from Kabi Vitrum and dispersed in sterile F 2 O under an inert atmosphere (N 2) under application of a high speed Waring ™ Blender for 2 minutes. The mixed material was transferred to a reservoir, again under inert atmosphere, which provided a Microfluidizer ™ Model 110 homogenizer. The material was circulated through the homogenizer for a total of 5 minutes at a flow rate of 350 ml / min using an air pressure of 4.22 kg / cm 2 to drive the pump piston, which corresponds to 562-633 kg / cm 2 at the homogenizer valve. The temperature was kept below 25 ° C at all times.

In this way, portions of approx. 750 g with concentrations in the range of 10 to 27%. The lecithin thus dispersed was collected under an inert atmosphere and stored at 4 ° C. All lecithin dispersions thus prepared were used within one week of their preparation.

Preparation of emulsion

The emulsifier reservoir was first filled with the appropriate amount of sterile water, glycerol and the lecithin dispersion described above. Next, the emulsifier was started and the oil was added through a syringe pump at a flow rate of 10 to 30 ml / minute directly into the homogenator's inlet port. Next, the perfluorocarbon was added at the same flow rate through a syringe pump through the same opening. The temperature 20 was constantly maintained at 45-48 ° C and the pH was maintained at 7.5-8.5 by controlled addition of 0.29 M NaHCO 3 or another base. For a 250 ml portion, the material was circulated through the homogenizer at a flow rate of 300-350 ml / minute for 10 minutes. Proportionally longer mean residence times were used for larger portions (e.g., a retention time of 20 minutes was used for a portion of 500 ml).

Analysis

Particle Size Distribution Analysis (PSD) 30 Samples were analyzed according to published process descriptions on a Brockhaven BI-90 Particle Sizer. All samples were prepared in 1 ml of isotonic glycerol solution immediately prior to analysis.

Serum Stability (PSD) 35 Samples were diluted 1: 1 with a 4: 1 mixture of Ringer's solution containing lactate and 25% human serum albumin (HSA), buffered to pH 7.2 with a phosphate buffer. Samples were incubated at 37 ° C for 120 hours before analyzing particle size distribution.

14 DK 172973 B1

Total replacement

Rats that had dual cannulas (neck veins) were subjected to isovolumetric replacement according to Goodin et al., "A Method for Evaluation of Blood Substitutes in the Conscious Animal," Am. dour. Physiol., 245, H519-523 (1983), until the hematocrit was ca. 3%. The original FiO2 of 0.8 was reduced daily by 0.1 over a period of 4 days, after which the animals were returned to room temperature.

Survival was determined after 14 days.

10 LD5Q.--inaljy.se

The test emulsion (50 ml / kg of a 4: 1 mixture of stock emulsion (40% by volume PFC) and support emulsion (equivalent to 16 ml / kg fluorocarbon component)) was infused at a rate of 1 ml / kg through the tail vein 15 of 10 Sprague-Dawley rats of 140-200 g under light ether anesthesia. After the infusion, the animals were returned to their cages and provided with feed and water ad libitum. The survival rate was determined after 14 days. Test emulsions with other PFC volume percentages would be prepared in a similar manner to feed 16 ml / kg of fluorocarbon component.

20

Laaerstabilitet

The test emulsions were stored in sealed 100 ml serum bottles under a nitrogen atmosphere at 4DC or 25 ° C for at least 3 months. After storage, the samples were tested for the distribution of particle size (laser light scattering), pH and visual quality (color, cream formation, etc.).

Specific Emulsions 47 different emulsions were made in duplicate by the methods outlined in the process section. Each emulsion contained perfluoro-decal in, tinted oil and egg yolk lecithin. The actual concentrations of the components of these emulsions are listed in Table I. Thereafter, the emulsions were analyzed as indicated above. The results are listed in Table II and plotted in FIG. 1 - unfilled circles for those who did not meet one or more requirements, and filled circles for those who met all requirements.

Table I

DK 172973 B1 15

Example Lecithin / PFD Color Threshold / PFD PFD% No. (w / w%) (w / w%) (w / w) 5 _ 1 0.66 2.6 30.0 2 1.15 13.0 30.0 3 2.08 17.4 30.0 4 2.60 0.7 30.0 10 5 2.60 2.6 30.0 6 2.60 5.2 30.0 7 3.99 10, 1 30.0 8 6.08 6.1 30.0 9 6.08 13.9 30.0 15 10 7.99 4.0 30.0 11 8.68 5.2 30.0 12 8.68 17 , 4 30.0 13 9.03 4.0 30.0 14 9.03 8.0 30.0 20 15 1.30 0.0 40.0 16 1.30 1.3 40.0 17 1.30 4.6 40.0 18 1.30 7.2 40.0 19 1.30 13.0 40.0 25 20 1.95 2.6 40.0 21 1.95 5.2 40.0 22 1, 95 7.8 40.0 23 1.95 10.4 40.0 24 2.60 2.6 40.0 30 25 2.93 2.6 40.0 26 2.93 5.2 40.0 27 2 , 93 7.8 40.0 28 2.93 10.4 40.0 29 2.91 1.3 40.0 35 30 3.91 3.9 40.0 31 3.91 6.5 40.0 32 3.91 10.4 40.0 33 4.60 1.3 40.0

Table I (cont. 16 DK 172973 B1 *)

Example Lecithin / PFD Color Threshold / PFD PFD% 5 No. (w / w%) (w / w%) (w / w) 34 4.60 13.0 40.0 35 5.20 0.0 40 , 0 36 5.99 3.9 40.0 10 37 7.81 0.0 40.0 38 7.81 1.3 40.0 39 7.81 7.2 40.0 40 7.81 13.0 40.0 41 9.11 2.6 40.0 15 42 7.81 2.6 50.0 43 2.60 2.6 50.0 44 0.66 2.6 50.0 45 2.60 0, 7 50.0 46 2.60 5.2 50.0 20 47 5.21 2.6 50.0

z Table II

DK 172973 B1 17

Example * PSD PSD LD ^ Survival 5 # (nM) (nM) (ml / kg) Total replacement

Serum (Supported) (120 h) 1,487,554 10 2,409,1262 3,285,564 4,269,267 5,221,230 6,235,242 15 7,205,223 8,270,245 9,280,276 10,279,254 11 290 290 20 12,474 429 13,388,377 14 388 320 15 363 460 16 290 436 90% 0% 25 17 269 427 18 314 1163 19 435 1592 70% 33% 20 221 366 21 219 30 22 245 400 23 269 916 24 207 327 95% 83% 25 178 319 26 172 35 27 213 410 28 202 511 29 277 274 30 220 247

Table II (continued! DK 172973 B1 18

Example 1 PSD PSD LD5Q Survival 5 No. (nM) (nM) (ml / kg) Total replacement

Serum (Supported) (120 hours) 10 31 259 255 32 327 511 33 239 205 34 555 705 35 235 15 36 320 287 37 219 38 379 306 - 100% 39 513 466 40 595 838 0% 67% 20 41 318 307 42 373 342 43 324 325 44 503 612 45 290 286 25 46 428 427 47 300 324

All emulsions (1-47) had a storage stability of more than 3 months at room temperature as measured by particle size distribution analysis and visual inspection.

Claims (21)

A physiologically acceptable emulsion, comprising a highly fluorinated organic compound, an oil which is not substantially surfactant and not substantially water-soluble, and a surfactant, characterized in that (a) the highly fluorinated organic compound is present in the emulsion in an amount between 20 and 60% by volume, and that 10 (b) the amounts of the surfactant and the oil depend on the volume percentage of the highly fluorinated organic compound and are present in amounts defined by that of FIG. 1 and the surfaces defining this volume and effectively producing 15 emulsions characterized by (1) a particle size distribution (PSD) of less than 400 nm after sterilization, (2) a serum stability characterized by a PSD of less than 400 nm after five days in serum or lone solutions, (3) an LD5Q value of 1 rat of at least 16 ml / kg of the highly fluorinated organic compound in the emulsion, (4) a survival rate of at least 70% at total replacement in 1 rat; and (5) a storage stability of at least several months at 4) C. Emulsion according to claim 1, characterized in that the highly fluorinated organic compound is present in an amount between 30 and 55% by volume. Emulsion according to claim 1, characterized in that the highly fluorinated organic compound is present in an amount of approx. 40% by volume. 30 Emulsion according to any one of claims 1 to 3, characterized in that the highly fluorinated organic compound is selected from perfluorodecaline and perfluorooctyl bromide. Emulsion according to claim 4, characterized in that the oil is selected from color-time oil and soybean oil. DK 172973 B1 20 Emulsion according to claim 5, characterized in that the surfactant is egg bioelecithin. The emulsion of claim 1, further comprising at least one compound selected from isotonic agents, osmotic pressure regulators, serum thrushing agents and antioxidants. A composition comprising an emulsion according to any one of claims 1 to 7. 10 A red blood cell replacement comprising an amount of an emulsion according to any one of claims 1 to 7, which is therapeutically effective for oxygen transport and delivery in humans. A biological imaging contrast agent comprising an amount of an emulsion according to any one of claims 1 to 7, which is clinically effective for imaging with modalities selected from nuclear magnetic resonance, X-ray, ultrasound and positron emission tomography. 20 A composition for improving radiation and chemotherapeutic treatment of cancer, comprising a therapeutically effective amount of an emulsion according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 25 A composition for minimizing the adverse effects of coronary balloon angioplasty, comprising a therapeutically effective amount of an emulsion according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 30 A composition for preserving organs, comprising a preservative amount of an emulsion according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. A composition for the treatment of heart attacks, strokes and vascular occlusions, comprising a therapeutically effective amount of an emulsion according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. DK 172973 B1 21 Use of an emulsion according to any one of claims 1 to 7 for the preparation of a red blood cell replacement to support the oxygen needs of living organisms. 5 Use of an emulsion according to any one of claims 1 to 7 for the preparation of a composition for improving radiation and chemotherapeutic treatment of cancer. Use of an emulsion according to any one of claims 1 to 7 for the preparation of a composition for minimizing the adverse effects of coronary balloon angioplasty in perfusion of the emulsion through the central lumen of a catheter. A method of preserving organs by perfusion of an emulsion according to any one of claims 1 to 7. The use of contrast agent according to claim 10 for the preparation of a composition for non-invasive imaging of internal organs and blood flow in connection with imaging by nuclear magnetic resonance, ultrasound, pos itron emission tomography or X-ray. Use of an emulsion according to any one of claims 1 to 7 for the preparation of a composition for the treatment of heart attacks, strokes and vascular occlusions. A physiologically acceptable emulsion comprising a highly fluorinated organic compound, an oil which is not substantially surfactant and not substantially water soluble, and a surfactant, characterized in that (a) the highly fluorinated organic compound is present in the emulsion 1 is an amount between 20 and 60% by volume, and that 35 (b) the amounts of the surfactant and oil depend on the volume percentage of the highly fluorinated organic compound and is present in amounts defined by that of FIG. 1 and the surfaces which define this volume and effectively produce 22 emulsions characterized by (1) a particle size distribution (PSD) of less than 300 nm after sterilization, (2) a serum stability characterized by a PSD of less than 300 after five days in serum or ion solutions, (3) an LD5Q value in rats of at least 5 16 ml / kg of the highly fluorinated organic compound in the emulsion, (4) a survival rate of at least 70% in total rats replacement, and (5) a storage stability of at least several months at 4) C. 10 15 20